Method and compositions for sequencing nucleic acid molecules

ABSTRACT

The invention relates to methods, compositions, kits and apparati for sequencing nucleic acid molecules. The invention particularly concerns the use of an exonuclease activity in concert with a polymerase activity to mediate such sequencing.

FIELD OF THE INVENTION

[0001] The invention relates to methods, compositions, kits and apparatifor sequencing nucleic acid molecules. The invention particularlyconcerns the use of an exonuclease activity in concert with a polymeraseactivity to mediate such sequencing.

BACKGROUND OF THE INVENTION

[0002] The capability of determining the sequences of nucleic acidmolecules is of fundamental importance to modern biology and medicine(Glasel J A. (2002) “DRUGS, THE HUMAN GENOME, AND INDIVIDUAL-BASEDMEDICINE,” Prog. Drug Res. 58:1-50; Green, E. D. (2001) “Strategies forthe Systemic Sequencing of Complex Genomes,” Nat. Rev. Genet. 2:6-12;Opalinska, J. B. et al. (2002) “NUCLEIC-ACID THERAPEUTICS: BASICPRINCIPLES AND RECENT APPLICATIONS,” Nat. Rev. Drug Discov. 1:503-514;Kim, Y. et al. (2002) “THE NUCLEOTIDE: DNA SEQUENCING AND ITS CLINICALAPPLICATION,” J. Oral Maxillofac. Surg. 60:924-930).

[0003] Initial attempts to determine the sequence of a DNA moleculeinvolved extensions of techniques that had been initially developed topermit the sequencing of RNA molecules (Sanger, F. (1965) “ATWO-DIMENSIONAL FRACTIONATION PROCEDURE FOR RADIOACTIVE NUCLEOTIDES,” J.Mol. Biol. 13:373-398; Brownlee, G. G. et al. (1968) “THE SEQUENCE OF 5S RIBOSOMAL RIBONUCLEIC ACID,” J. Molec. Biol. 34:379-412). Such methodsexploited the specific cleavage of DNA into smaller fragments by (1)enzymatic digestion (Robertson, H. D. et al. (1973) “ISOLATION ANDSEQUENCE ANALYSIS OF A RIBOSOME-PROTECTED FRAGMENT FROM BACTERIOPHAGEΦX174 DNA,” Nature New Biol. 241:38-40; Ziff, E. B. et al. (1973)“DETERMINATION OF THE NUCLEOTIDE SEQUENCE OF A FRAGMENT OF BACTERIOPHAGEΦX174 DNA,” Nature New Biol. 241:34-37); (2) nearest neighbor analysis(Wu, R. et al. (1971) “Nucleotide Sequence Analysis Of DNA. 1′. CompleteNucleotide Sequence Of The Cohesive Ends Of Bacteriophage Lambda DNA,”J. Molec. Biol. 57:491-511), or (3) the “Wandering SPOT” method (Sanger,F. (1973) “USE OF DNA POLYMERASE I PRIMED BY A SYNTHETIC OLIGONUCLEOTIDETO DETERMINE A NUCLEOTIDE SEQUENCE IN PHAGE FL DNA,” Proc. Natl. Acad.Sci. (U.S.A.) 70:1209-1213 (1973).

[0004] The most commonly used methods of nucleic acid sequencingcomprise the “dideoxy-mediated chain termination method,” also known asthe “Sanger Method” (Sanger, F. et al. (1975) “A RAPID METHOD FORDETERMINING SEQUENCES IN DNA BY PRIMED SYNTHESIS WITH DNA POLYMERASE,”J. Molec. Biol. 94:441-448 (1975); Sanger, F. et al. (1977) “DNASEQUENCING WITH CHAIN-TERMINATING INHIBITORS,” Proc. Natl. Acad. Sci.(USA) 74:5463-5467; Prober, J. et al. “A SYSTEM FOR RAPID DNA SEQUENCINGWITH FLUORESCENT CHAIN-TERMINATING DIDEOXYNUCLEOTIDES,” (1987) Science238:336-341 (1987)) and the “chemical degradation method,” also known asthe “Maxam-Gilbert method” (Maxam, A. M. et al. (1977) “NEW METHOD FORSEQUENCING DNA.,” Proc. Natl. Acad. Sci. (U.S.A.) 74:560-564). Methodsfor sequencing DNA using either the dideoxy-mediated method or theMaxam-Gilbert method are widely known to those of ordinary skill in theart. Such methods are, for example, disclosed in Maniatis, T. et al.(1989) “MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Edition,” ColdSpring Harbor Press, Cold Spring Harbor, N.Y., and in Zyskind, J. W. etal. (1988) RECOMBINANT DNA LABORATORY MANUAL, Academic Press, Inc., NewYork. Methods of DNA sequencing are reviewed by Marziali, A. et al.(2001) (“NEW DNA SEQUENCING METHODS,” Ann. Rev. Biomed. Eng. 3:195-223),Graham, C. A. et al. (2001) (“INTRODUCTION TO DNA SEQUENCING,” MethodsMolec. Biol. 167:1-12), Messing, J. (2001) (“THE UNIVERSAL PRIMERS ANDTHE SHOTGUN DNA SEQUENCING METHOD,” Methods Molec. Biol. 167:13-31) andBankier, A. T. (2001) (“SHOTGUN DNA SEQUENCING,” Methods Molec. Biol.167:89-100).

[0005] The Maxam-Gilbert method of DNA sequencing is a degradativemethod in which a fragment of DNA is labeled at one end (or terminus)and partially cleaved in four separate chemical reactions, each of whichis specific for cleaving the DNA molecule at a particular base (G or C)at a particular type of base (A/G, C/T, or A>C). The effect of suchreactions is to create a set of nested molecules whose lengths aredetermined by the locations of a particular base along the length of theDNA molecule being sequenced. The nested reaction products are thenresolved by electrophoresis, and the end-labeled molecules are detected,typically by autoradiography when a ³²P label is employed. Four singlelanes are typically required in order to determine the sequence.Although the Maxam-Gilbert method uses simple chemical reagents whichare readily available, it is extremely laborious to perform and requiresmeticulous experimental technique.

[0006] Owing to these deficiencies, the dideoxy-mediated or “Sanger”chain termination method of DNA sequencing has become the method ofchoice. In the dideoxy-mediated sequencing method, the sequence of a DNAmolecule is obtained through the extension of an oligonucleotide primerthat is hybridized to the nucleic acid molecule being sequenced. Inbrief, four separate primer extension reactions are conducted. In eachreaction, a DNA polymerase is added along with the four nucleotidetriphosphates (dATP, dCTP, dGTP, and dTTP) needed to polymerize DNA.Significantly, each reaction also contains a 2′,3′ dideoxy derivative ofthe dATP, dCTP, dGTP, or dTTP nucleotides. Such derivatives differ fromconventional nucleotides in lacking a hydroxyl residue at the 3′position of deoxyribose. Although DNA polymerases can incorporate adideoxy nucleotide into the primer extension product, such incorporationblocks further primer extension. Thus, the incorporation of a dideoxyderivative results in the termination of the extension reaction.

[0007] By conducting the dideoxy sequencing reaction under conditions inwhich the dideoxy nucleotides are present in lower concentrations thantheir corresponding conventional nucleotides, the net result of each ofthe four reactions is the production of a nested set ofoligonucleotides, each of which is terminated by the particular dideoxyderivative used in the reaction. By subjecting the reaction products ofeach of the extension reactions to electrophoresis, it is possible toobtain a series of four “ladders,” of bands. Since the position of each“rung” of the ladder is determined by the size of the molecule, andsince such size is determined by the incorporation of the dideoxyderivative, the appearance and location of a particular “rung” can bereadily translated into the sequence of the extended primer. Thus, thesequence of the extended primer can be determined throughelectrophoretic analysis.

[0008] The adoption of the Sanger method as the method of choice wasspurred by the development of novel polymerases that could more readilyincorporate fluorescent and other non-radioactively labeleddideoxynucleotides (Tabor, S. et al. (1995) “A SINGLE RESIDUE IN DNAPOLYMERASES OF THE E SCHERICHIA C OLI DNA POLYMERASE I FAMILY ISCRITICAL FOR DISTINGUISHING BETWEEN DEOXY- AND DIDEOXYRIBONUCLEOTIDES,”Proc. Natl. Acad. Sci. USA 92, 6339-6343; Tabor, S. et al. (U.S. Pat.No. 5,614,365, U.S. Pat. No. 5,674,716).

[0009] As originally implemented, the “Sanger” method required separatesequencing reactions for each of the four possible nucleotides. Onealternative to this requirement was developed by Prober, J. M. et al.,who developed differentially labeled dideoxynucleoside triphosphates.The use of such reagents enables the sequencing reaction to be conductedin a single reaction tube (Prober, J. M. et al. (1987) “A SYSTEM FORRAPID DNA SEQUENCING WITH FLUORESCENT CHAIN-TERMINATINGDIDEOXYNUCLEOTIDES,” Science 238:336-341; Prober, et al. (U.S. Pat. No.5,242,796); Prober, et al. (U.S. Pat. No. 5,306,618); Prober, et al.(U.S. Pat. No. 5,332,666); Lee, L. G. et al. (1992) discloses the use ofdye-labeled terminators, and their incorporation into DNA by T7polymerase (Lee, L. G. et al. (1992) “DNA SEQUENCING WITH DYE-LABELEDTERMINATORS AND T7 DNA POLYMERASE: EFFECT OF DYES AND DNTPs ONINCORPORATION OF DYE-TERMINATORS AND PROBABILITY ANALYSIS OF TERMINATIONFRAGMENTS,” Nucl. Acids Res. 20:2471-2483).

[0010] An essential characteristic of the “Sanger” method is theinclusion of conventional nucleotides and chain-terminator nucleotidesin the same sequencing reaction. The inclusion of such a combination ofnucleotide species is necessary in order to form the nested set ofprimer extension molecules that is required by the method. A variety of“microsequencing” methods have, however, been developed that employfewer than all four conventional nucleotides, or that employ subsets ofconventional and/or chain terminator nucleotide species. Such methodsare employed in sequencing single nucleotide polymorphisms, and inconjunction with the use of random or pseudo-random ordered arrays ofoligonucleotides.

[0011] For example, some such methods rely on the incorporation oflabeled deoxynucleotides to discriminate between bases at a polymorphicsite (Kornher, J. S. et al. (1989) “MUTATION DETECTION USING NUCLEOTIDEANALOGS THAT ALTER ELECTROPHORETIC MOBILITY,” Nucl. Acids Res.17:7779-7784; Sokolov, B. P. (1990) “PRIMER EXTENSION TECHNIQUE FOR THEDETECTION OF SINGLE NUCLEOTIDE IN GENOMIC DNA,” Nucl. Acids Res.18:3671; Syvanen, A.-C., et al. (1990) “A PRIMER-GUIDED NUCLEOTIDEINCORPORATION ASSAY IN THE GENOTYPING OF APOLIPOPROTEIN E,” Genomics8:684-692; Bajaj et al. (U.S. Pat. No. 5,846,710); Kuppuswamy, M. N. etal. (1991) “SINGLE NUCLEOTIDE PRIMER EXTENSION TO DETECT GENETICDISEASES: EXPERIMENTAL APPLICATION TO HEMOPHILLIA B (FACTOR IX) ANDCYSTIC FIBROSIS GENES,” Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147;Prezant, T. R. et al. (1992) “TRAPPED-OLIGONUCLEOTIDE NUCLEOTIDEINCORPORATION (TONI) ASSAY, A SIMPLE METHOD FOR SCREENING POINTMUTATIONS,” Hum. Mutat. 1: 159-164; Ugozzoli, L. et al. (1992)“DETECTION OF SPECIFIC ALLELES BY USING ALLELE-SPECIFIC PRIMER EXTENSIONFOLLOWED BY CAPTURE ON SOLID SUPPORT,” GATA 9:107-112; Nyren, P. et al.(1993) “SOLID PHASE DNA MINISEQUENCING BY AN ENZYMATIC LUMINOMETRICINORGANIC PYROPHOSPHATE DETECTION ASSAY,” Anal. Biochem. 208:171-175;and Wallace (WO89/10414). Alternate methods involve combinations ofconventional and chain-terminating nucleotides (Syvanen, A.-C. et al.(1993) “IDENTIFICATION OF INDIVIDUALS BY ANALYSIS OF BIALLELIC DNAMARKERS, USING PCR AND SOLID-PHASE MINISEQUENCING,” Amer. J. Hum. Genet.52:46-59 (1993); Soderlund et al. (U.S. Pat. No. 6,013,431); Kornher, J.S. et al. (1989) “MUTATION DETECTION USING NUCLEOTIDE ANALOGS THAT ALTERELECTROPHORETIC MOBILITY,” Nucl. Acids Res. 17:7779-7784). Other methodsrequire the presence of chain-terminator nucleotides and the absence ofconventional nucleotides (Goelet, P. et al. (WO 92/15712, U.S. Pat. No.6,004,744, U.S. Pat. No. 5,952,174, U.S. Pat. No. 5,888,819).

[0012] Goelet, P. et al. (U.S. Pat. No. 5,888,819), for example,concerns a method for determining the identity of a nucleotide base at aspecific position in a nucleic acid of interest in which a samplecontaining the nucleic acid of interest, in single-stranded form, iscontacted with an oligonucleotide primer that is fully complementary toand which hybridizes specifically to a stretch of nucleotide bases ofthe nucleic acid of interest immediately adjacent to the nucleotide baseto be identified, under high stringency hybridization conditions, so asto form a double-stranded nucleic acid molecule in which the nucleotidebase to be identified is the first unpaired base in the templateimmediately downstream of the 3′ end of the primer. The double-strandedmolecule is incubated, in the absence of non-chain terminatornucleotides, with at least two different chain terminator nucleotides,and in the presence of a polymerase, under conditions sufficient tocause a template-dependent, primer extension reaction to occur that isstrictly dependent upon the identity of the unpaired nucleotide base inthe template immediately downstream of the 3′ end of the primer. Theidentity of the nucleotide base to be identified is determined bydetecting the identity of the incorporated chain-terminator nucleotide.

[0013] Mundy, C. R. (U.S. Pat. No. 4,656,127) discusses an alternativemicrosequencing method that employs a specialized exonuclease resistantnucleotide derivative. A primer complementary to an allelic sequenceimmediately 3′-to the polymorphic site is permitted to hybridize to atarget molecule obtained from a particular animal or human. If thepolymorphic site on the target molecule contains a nucleotide that iscomplementary to the particular exonucleotide-resistant nucleotidederivative present, then that derivative will be incorporated by apolymerase onto the end of the hybridized primer. Such incorporationrenders the primer resistant to exonuclease, and thereby permits itsdetection. Since the identity of the exonucleotide-resistant derivativeof the sample is known, a finding that the primer has become resistantto exonucleases reveals that the nucleotide present in the polymorphicsite of the target molecule was complementary to that of the nucleotidederivative used in the reaction. Mundy's method has the advantage thatit does not require the determination of large amounts of extraneoussequence data. It has the disadvantages of destroying the amplifiedtarget sequences, and unmodified primer and of being extremely sensitiveto the rate of polymerase incorporation of the specific exonucleaseresistant nucleotide being used.

[0014] Cohen, D. et al. (French Patent 2,650,840; WO91/02087) discuss asolution-based method for determining the identity of the nucleotide ofa polymorphic site. As in the method of Mundy (U.S. Pat. No. 4,656,127),a primer is employed that is complementary to allelic sequencesimmediately 3′-to a polymorphic site. The method determines the identityof the nucleotide of that site using labeled dideoxynucleotidederivatives, which, if complementary to the nucleotide of thepolymorphic site will become incorporated onto the terminus of theprimer. Cheesman, P. (U.S. Pat. No. 5,302,509) describes a method forsequencing a single stranded DNA molecule using fluorescently labeled3′-blocked nucleotide triphosphates. An apparatus for the separation,concentration and detection of a DNA molecule in a liquid sample hasbeen recently described by Ritterband, et al. (PCT Patent ApplicationNo. WO95/17676). Dower, W. J. et al. (U.S. Pat. No. 5,547,839) describesa method for sequencing an immobilized primer using fluorescent labels.Chee, M. et al. (WO95/11995) describes an array of primers immobilizedonto a solid surface. Chee et al. further describes a method fordetermining the presence of a mutation in a target sequence by comparingagainst a reference sequence with a known sequence.

[0015] In a further variation of such methods, ordered arrays ofsolid-phase bound random or pseudorandom oligonucleotides to function asprimers for the sequencing reaction. In brief, such methods avoid theneed for obtaining nested sets of fragments by hybridizing the intactmolecule being sequenced with an array of solid-phase bound primers ofknown sequence and position. The reaction is conducted in the presenceof labeled nucleotides or labeled dideoxy nucleotides (Chetverin, A. B.et al. (1994) “OLIGONUCLEOTIDE ARRAYS: NEW CONCEPTS AND POSSIBITIIES,”Bio/Technology 12:1093-1099; Macevicz (U.S. Pat. No. 5,002,867);Beattie, W. G. et al. (1995) “HYBRIDIZATION OF DNA TARGETS TOGLASS-TETHERED OLIGONUCLEOTIDE PROBES,” Molec. Biotech. 4:213-225;Boyce-Jacino et al. (U.S. Pat. No. 6,294,336); Head et al. (U.S. Pat.No. 6,322,968); Head et al. (U.S. Pat. No. 6,337,188). Caskey, C. et al.has described a method of analyzing a polynucleotide of interest usingone or more sets of consecutive oligonucleotide primers differing withineach set by one base at the growing end thereof (Caskey, C. et al. (WO95/00669)). The oligonucleotide primers are extended with a chainterminating nucleotide and the identity of each terminating nucleotideis determined.

[0016] Pastinen, T. et al. has described a method for the multiplexdetection of mutations wherein the mutations are detected by extendingimmobilized primers, that anneal to the template sequences immediatelyadjacent to the mutant nucleotide positions, with a single labeleddideoxynucleotide using a DNA polymerase (Pastinen, T. et al. (1997)“MINISEQUENCING: A SPECIFIC TOOL FOR DNA ANALYSIS AND DIAGNOSTICS ONOLIGONUCLEOTIDE ARRAYS,” Genome Res. 7:606-614). In this method, theoligonucleotide arrays were prepared by coupling one primer per mutationto be detected on a small glass area. Pastinen, T. et al. has alsodescribed a method to detect multiple single nucleotide polymorphisms inan undivided sample (Pastinen, T. et al. (1996) “MULTIPLEX, FLUORESCENT,SOLID-PHASE MINISEQUENCING FOR EFFICIENT SCREENING OF DNA SEQUENCEVARIATION,” Clin. Chem. 42:1319-1397). According to this method, theamplified DNA templates are first captured onto a manifold and then,with multiple minsequencing primers, single nucleotide extensionreactions are carried out simultaneously with fluorescently labeleddideoxynucleotides.

[0017] Jalanko, A. et al. has described the application of solid-phaseminisequencing methods to the detection of a mutation causing cysticfibrosis (Jalanko, A. et al. (1992) “SCREENING FOR DEFINED CYSTICFIBROSIS MUTATIONS BY SOLID-PHASE MINISEQUENCING,” Clin. Chem.38:39-43). In this method, an amplified DNA molecule that isbiotinylated at its 5′ terminus is bound to a solid phase and denatured.A detection primer, which hybridizes immediately before the putativemutation, is hybridized to the immobilized single stranded template andelongated with a single, labeled deoxynucleoside residue. Shumaker, J.M. et al. has described another solid phase primer extension method formutation detection (Shumaker, J. M. et al. “MUTATION DETECTION BY SOLIDPHASE PRIMER EXTENSION,” (1996) Hum. Mutation 7:346-354). In thismethod, template DNA is annealed to an oligonucleotide array, extendedwith ³²P dNTPs and analyzed with a phosphoimager.

[0018] Sequencing determination methods have also been developed thatrely on the extent of hybridization between a probe and a templatemolecule (Drmanac, R. et al. (2002) “SEQUENCING BY HYBRIDIZATION (SBH):ADVANTAGES, ACHIEVEMENTS, AND OPPORTUNITIES,” Adv. Biochem. Eng.Biotechnol. 77:75-101; Drmanac, R. et al. (2001) “SEQUENCING BYHYBRIDIZATION ARRAYS,” Methods Molec. Biol. 170:39-51; Gabig, M. et al.(2001) “AN INTRODUCTION TO DNA CHIPS: PRINCIPLES, TECHNOLOGY,APPLICATIONS AND ANALYSIS,” Acta Biochim. Pol. 48:615-22). Drmanac, R.T., for example, has described a method for sequencing nucleic acid byhybridization using nucleic acid segments on different sectors of asubstrate and probes that discriminate between a one base mismatch(Drmanac, R. T. (EP 797683)). Gruber, L. S. has described a method forscreening a sample for the presence of an unknown sequence usinghybridization sequencing (Gruber, L. S. (EP 787183)). Landegren, U. etal. have described the “Oligonucleotide Ligation Assay” (“OLA”)(Landegren, U. et al. (1988) “LIGASE-MEDIATED GENE DETECTION TECHNIQUE,”Science 241:1077-1080) as being capable of detecting single nucleotidepolymorphisms. The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is biotinylated, and theother is detectably labeled. If the precise complementary sequence isfound in a target molecule, the oligonucleotides will hybridize suchthat their termini abut, and create a ligation substrate. Ligation thenpermits the labeled oligonucleotide to be recovered using avidin, oranother biotin ligand. Nickerson, D. A. et al. have described a nucleicacid detection assay that combines attributes of the polymerase chainreaction (PCR) and OLA (Nickerson, D. A. et al. (1990) “AUTOMATED DNADIAGNOSTICS USING AN ELISA-BASED OLIGONUCLEOTIDE LIGATION ASSAY,” Proc.Natl. Acad. Sci. (U.S.A.) 87:8923-8927). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA. In addition to requiring multiple, and separateprocessing steps, one problem associated with such combinations is thatthey inherit all of the problems associated with PCR and OLA.

[0019] Exonucleases are enzymes that degrade nucleic acid molecules fromeither their 3′ or 5′ terminus. As indicated above, exonucleases havebeen used to facilitate DNA sequencing (Mundy, C. R. (U.S. Pat. No.4,656,127)). Jett et al. have proposed the use of exonucleases toaccomplish the stepwise degradation of a target nucleic acid moleculeand the sequential analysis of each, released nucleotide (Jett, J. H. etal. (1989) “HIGH-SPEED DNA SEQUENCING: AN APPROACH BASED UPONFLUORESCENCE DETECTION OF SINGLE MOLECULES,” J Biomolecular Structure &Dynamics 7:301-309; Jett et al. (WO 89/03432)). Koster (U.S. Pat. No.6,140,053; U.S. Pat. No. 6,074,823) disclose a sequencing strategy thatuses mass spectroscopy to analyze the differences in mass of thefragments obtained through exonuclease digestion. Murtagh (U.S. Pat. No.5,688,669) describes the use of the 3′ to 5′ exonuclease, ExonucleaseIII, to digest a target DNA molecule in to fragments and then determinetheir sequence via hybridization to complementary probes.

[0020] Labeit, S. et al. have disclosed a sequencing method in whichfour separate primer extension reactions are conducted, each in thepresence of a different phosphothioated deoxynucleoside and threeconventional nucleotides (Labeit, S. et al. “LABORATORY METHODS, A NEWMETHOD OF DNA SEQUENCING USING DEOXYNUCLEOSIDE A-TRIPHOSPHATES,” DNA4:173-177). The primer extension reactions are then incubated in thepresence of Exonuclease III. Since exonucleases cannot cleavephosphothioated nucleotides, treatment with the exonuclease results inthe production of a nested set of fragments each containing aphosphothioated nucleoside at its 3′ terminus (Putney, S. D. et al.(1981) “A DNA FRAGMENT WITH AN ALPHA-PHOSPHOROTHIOATE NUCLEOTIDE AT ONEEND IS ASYMMETRICALLY BLOCKED FROM DIGESTION BY EXONUCLEASE III AND CANBE REPLICATED IN VIVO,” Proc Natl Acad Sci (USA) 78:7350-7354; Nakamaye,K. L. et al. (1988) “DIRECT SEQUENCING OF POLYMERASE CHAIN REACTIONAMPLIFIED DNA FRAGMENTS THROUGH THE INCORPORATION OF DEOXYNUCLEOSIDEα-THIOTRIPOSPHATES,” Nucleic Acids Res. 16:9947-9959). The sequences ofthe molecules can be determined using gel electrophoresis methods.

[0021] Iyyalasomayazula (U.S. Pat. No. 6,165,726) describes the biotinlabeling of molecules for sequencing, and the use of immobilizedstreptavidin to capture such molecules.

[0022] Despite the development of all such methods, a need continues toexist for an improved, rapid, and sensitive method for sequencing DNAthat avoids the need for specialized enzymes and procedures. The presentinvention is directed to this and other goals.

SUMMARY OF THE INVENTION

[0023] Current DNA sequencing strategies revolve around the use ofprimer extension on a target or template DNA. In the most widelyemployed method, all four deoxynucleotides are included in thepolymerization reaction as well as 4 dideoxynucleotides. Since automatedDNA sequencers have the ability to distinguish different dyes, acomplete sequencing reaction can be performed in a single tube if eachdideoxynucleotide is labeled with a different dye. The resulting DNAfragments are the products of primer extensions from a single primer,but termination results from the incorporation of 4 different dyelabeled dideoxynucleotides. The present invention is intended to providean alternative sequencing method, and, in preferred embodiments produces4 different dye labeled DNA fragments by a novel approach that employsmore robust chemistries and involves less stringent requirements for“special” polymerases

[0024] In detail, the invention provides a method for determining thesequence of a region of one strand of a double-stranded nucleic acidtarget molecule, wherein the method comprises incubating the nucleicacid target molecule in the presence of an exonuclease activity, apolymerase activity and four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species.

[0025] The invention particularly concerns the embodiment of suchmethods wherein four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species are employed.The invention further concerns the embodiment of such methods wherein atleast one of the four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species is fluorescentlylabeled. The invention further concerns the embodiment of such methodswherein the four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species arefluorescently labeled.

[0026] The invention further concerns the embodiment of such methodswherein the double-stranded nucleic acid target molecule possesses onlyone 3′ terminus that is a substrate for the exonuclease activity. Theinvention further concerns the embodiment of such methods wherein thedouble-stranded nucleic acid target molecule possesses a 3′ terminusthat extends beyond the 5′ terminus of the opposite strand. Theinvention further concerns the embodiment of such methods wherein thedouble-stranded nucleic acid target molecule possesses a 3′ terminusthat is sterically blocked from exonuclease activity degradation. Theinvention additionally concerns the embodiment of such methods whereinboth strands of the double-stranded nucleic acid target molecule possessa 3′ terminus that is a substrate for the exonuclease activity.

[0027] The invention further concerns the embodiments of such methodswherein one 5′ terminus or both 5′ termini of the double-strandednucleic acid target molecule possesses a haptenic group, especiallywherein the haptenic group is biotin.

[0028] The invention further concerns a method for determining thenucleotide sequence of a region of a double-stranded nucleic acid targetmolecule, wherein the method comprises the steps:

[0029] (A) incubating a preparation of the double-stranded targetmolecule in the presence of a 3′ to 5′ exonuclease activity, wherein thedouble-stranded nucleic acid target molecule possess at least one 3′terminus that is a substrate for the exonuclease activity, wherein theincubation is conducted under conditions sufficient to permit theexonuclease activity to produce a nested population of double-strandednucleic acid target molecule having at least one degraded 3′ termini;

[0030] (B) incubating the nested population of double-stranded nucleicacid target molecule in the presence of a polymerase activity and atleast one detectably labeled, exonuclease activity-resistant, chainterminator nucleotide species, wherein the incubation is conducted underconditions sufficient to permit the polymerase activity to mediate thetemplate-dependent incorporation of one of the nucleotide species ontothe 3′ terminus of a nucleic acid target molecule whose 3′ terminus wasdegraded by the exonuclease activity; and

[0031] (C) determining the identity of the differentially detectable,exonuclease activity-resistant, chain terminator nucleotide speciesincorporated onto the 3′ terminus at the selected region.

[0032] The invention further concerns the embodiment of such methodwherein the steps A and B are conducted simultaneously, and wherein theconditions employed are sufficient to permit the exonuclease activity todegrade the substrate termini and sufficient to permit the polymeraseactivity to mediate the template-dependent incorporation of thenucleotide species.

[0033] The invention particularly concerns the embodiment of suchmethods wherein four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species are employed.The invention further concerns the embodiment of such methods wherein atleast one of the four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species is fluorescentlylabeled. The invention further concerns the embodiment of such methodswherein the four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species arefluorescently labeled.

[0034] The invention further concerns the embodiment of such methodswherein the double-stranded nucleic acid target molecule possesses onlyone 3′ terminus that is a substrate for the exonuclease activity. Theinvention further concerns the embodiment of such methods wherein thedouble-stranded nucleic acid target molecule possesses a 3′ terminusthat extends beyond the 5′ terminus of the opposite strand. Theinvention further concerns the embodiment of such methods wherein thedouble-stranded nucleic acid target molecule possesses a 3′ terminusthat is sterically blocked from exonuclease activity degradation. Theinvention additionally concerns the embodiment of such methods whereinboth strands of the double-stranded nucleic acid target molecule possessa 3′ terminus that is a substrate for the exonuclease activity.

[0035] The invention further concerns the embodiments of such methodswherein one 5′ terminus or both 5′ termini of the double-strandednucleic acid target molecule possesses a haptenic group, especiallywherein the haptenic group is biotin.

[0036] The invention also concerns an in vitro composition comprising adouble-stranded nucleic acid target molecule, an exonuclease activity, apolymerase activity and four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species.

[0037] The invention further concerns the embodiments of suchcomposition wherein at least one of the four differentially detectable,exonuclease activity-resistant, chain terminator nucleotide species isfluorescently labeled. The invention further concerns the embodiments ofsuch compositions wherein the four differentially detectable,exonuclease activity-resistant, chain terminator nucleotide species arefluorescently labeled. The invention further concerns the embodiments ofsuch compositions wherein at least one 5′ terminus or wherein both 5′termini of the double-stranded nucleic acid target molecule possesses ahaptenic group, especially wherein the haptenic group is biotin.

[0038] The invention further concerns a composition comprising fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species. The invention further concerns theembodiments of such composition wherein at least one of the fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species is fluorescently labeled. The inventionfurther concerns the embodiments of such compositions wherein the fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species are fluorescently labeled.

[0039] The invention further concerns a kit specially adapted tofacilitate the sequencing of a target nucleic acid molecule, the kitcomprising a first container comprising a primer A, a second containercomprising a primer B, and a third container containing an exonucleaseactivity, wherein the primers A and B mediate the amplification of adouble-stranded nucleic acid molecule comprising the target nucleic acidmolecule, and wherein at least one of the primer A or the primer Bpossesses a 5′ terminus having at least one modified nucleotide.

[0040] The invention further concerns the embodiments of such kitwherein the modified nucleotide is a ribonucleotide, a dUridinenucleotide, a phosphothioate nucleotide, or a biotin-derivatizednucleotide. The invention further concerns the embodiments of such kitswherein the kit further comprises a fourth container containing fourdetectably labeled, exonuclease activity-resistant, chain terminatornucleotide species, and especially wherein the four detectably labeled,exonuclease activity-resistant, chain terminator nucleotide species arefluorescently labeled.

[0041] The invention further concerns a sequenator, comprising anapparatus for determining the identity of fluoresecently labeledexonuclease activity-resistant, chain terminator nucleotide speciesincorporated onto the 3′ termini of a nucleic acid target molecule whose3′ terminus was degraded by the exonuclease; and then extended by atemplate-dependent polymerase to incorporate the fluorescently labelednucleotide species.

BRIEF DESCRIPTION OF THE FIGURES

[0042]FIG. 1 illustrates the use of a preferred embodiment of theinvention to sequence double-stranded DNA. In the figure, B representsBiotin; closed solid circles, striped circles, open circles, anddot-filled circles represent four differentially detectable exonucleaseactivity-resistant, chain terminator nucleotide species.

[0043]FIG. 2 illustrates the use of the present invention to sequenceone or both strands of a double-stranded nucleic acid target molecule.In the figure closed solid circles, striped circles, open circles, anddot-filled circles represent four differentially detectable exonucleaseactivity-resistant, chain terminator nucleotide species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The invention relates to methods, compositions, kits and apparatifor sequencing nucleic acid molecules, including RNA or DNA. Theinvention particularly concerns the incubation of reagents in thepresence of exonuclease activity, especially in concert with apolymerase activity, in order to mediate such sequencing.

[0045] The term “exonuclease activity,” as used herein refers to anenzymatic activity (or a chemical process equivalent thereof) that iscapable of removing a nucleotide from the terminus of a nucleic acidmolecule. Preferred exonuclease activities can remove nucleotides fromthe 3′ termini of a nucleic acid molecule. Examples of such preferred 3′to 5′ exonuclease activities include the 3′ to 5′ exonuclease activityof snake venom phosphodiesterase, the 3′ to 5′ exonuclease activity ofspleen phosphodiesterase, the 3′ to 5′ exonuclease activity of Bal-31nuclease, the 3′ to 5′ exonuclease activity of E. coli exonuclease I,the 3′ to 5′ exonuclease activity of E. coli exonuclease VII, the 3′ to5′ exonuclease activity of Mung Bean Nuclease, the 3′ to 5′ exonucleaseactivity of S1 Nuclease, the 3′ to 5′ exonuclease activity of E. coliDNA polymerase I, the 3′ to 5′ exonuclease activity of the Klenowfragment of DNA polymerase I, the 3′ to 5′ exonuclease activity of T4DNA polymerase, the 3′ to 5′ exonuclease activity of T7 DNA polymerase,the 3′ to 5′ exonuclease activity of E. coli exonuclease III, the 3′ to5′ exonuclease activity of k exonuclease, the 3′ to 5′ exonucleaseactivity of Pyrococcus species GB-D DNA polymerase and the 3′ to 5′exonuclease activity of Thermococcus litoralis DNA polymerase. E. coliexonuclease III is particularly preferred for use in the presentinvention.

[0046] As used herein, the term “polymerase activity” refers to anenzymatic activity (or a chemical process equivalent thereof) that iscapable of extending the terminus of a nucleic acid molecule in atemplate-dependent manner (e.g., by mediating the incorporation of anucleotide onto the 3′ terminus of a primer molecule hybridized to acomplementary template). Polymerase activities relevant to the presentinvention include the polymerase activity of thermostable polymerases(such as Accuzyme, Biolase Diamond polymerase (Bioline); Tbr Polymerase,Tfl polymerase, Tsp B polymerase (BioNexus; www.bionexus.net); Thermuspolymerase (Chimerx; www.chimerx.com); MasterAmp Amplitherm polymerase,MasterAmp Tfl polymerase (Epicentre; www.epicentre.com); DyN/Azyme I andII polymerase (Finnzymes; www.finnzymes.com); Accutherm polymerase(GeneCraft; www.genecraft.de); Taq polymerase, ThermalAce polymerase(Invitrogen; www.invitrogen.com); VentR (exo-) polymerase, VentRpolymerase, Deep VentR (exo-) polymerase, Deep VentR polymerase, Bstpolymerase (New England Biolabs; www.neb.com); Pfu Polymerase, TflPolymerase, Tli Polymerase (Promega; www.promega.com); Pyra exo(-)polymerase, Tfu Polymerase (Qbiogene; www.qbiogene.com); Tgo Polymerase,Pwo Polymerase (Roche Molecular Biochemicals; biochem.roche.com); Pfunative polymerase, Pfu recombinant polymerase, PfuTurbo polymerase(Stratagene; www.stratagene.com); Pwo polymerase (ThermoHybaid;www.thermohybaid.com), etc., as well as the polymerase activity ofnon-thermostable polymerases (such as DNA polymerase III from E. coli,Klenow polymerase, T4 polymerase, T7 polymerase, Φ29 polymerase, etc.).

[0047] In accordance with the principles of the present invention,suitable polymerase activities are possessed by polymerases that areable to mediate the incorporation into nucleic acid molecules ofnucleotides and nucleotide analogs that are not substrates ofexonuclease activity. Preferably, such polymerase activities will becapable of mediating the incorporation of modified nucleotides (e.g.,methylated nucleotides, phosphothioated nucleotides, ribonucleotides),and especially chain terminator nucleotide species (such asdideoxynucleotides), and/or labeled nucleotides (such as thosepossessing fluorescent (e.g., α-thio dye terminators, borate dyeterminators, etc.), radioactive, paramagnetic, chemiluminescent,enzymatic, haptenic, antigenic, etc., labels.

[0048] In preferred embodiments, the invention is directed to a methodfor sequencing nucleic acid molecules in which the individual moleculesof a preparation of target molecules is subjected to 3′ exonucleaseactivity-mediated digestion, and to polymerase activity-mediatedextension in the presence of exonuclease activity-resistantchain-terminating nucleotides or nucleotide derivatives. The inventioncontemplates that the exonuclease activity treatment may precede, or maybe accomplished simultaneously with, the polymerase activity-mediatedextension reaction.

[0049] As indicated above, the preferred embodiments of the presentinvention employs differentially detectable, exonucleaseactivity-resistant, chain-terminating nucleotides or nucleotidederivatives. Any modification that renders the incorporated nucleotide“chain terminating” may be employed. Particularly preferred are thedideoxynucleotides whose ribosyl moiety lacks a 3′ hydroxyl group.

[0050] Depending upon the desired application, one, two, three or fourdifferent exonuclease activity-resistant chain-terminating nucleotidesor nucleotide derivatives may be employed. For example, determinationsof single nucleotide polymorphisms may be accomplished using one, two,three or four different exonuclease activity-resistant chain-terminatingnucleotides or nucleotide derivatives. Applications involving thesequencing of DNA, will preferably entail the use of four differentexonuclease activity-resistant chain-terminating nucleotides ornucleotide derivatives will be employed.

[0051] Preferably, the employed exonuclease activity-resistantchain-terminating nucleotides will be differentially detectable. As usedherein, the term “differentially detectable” denotes the use or presenceof a label that that can be detected even in the presence of anotherlabel. Such differentially detectability can be attained in a variety ofways. For example, different classes of labels (e.g., some radioactive,some fluorescent, etc.) may be used. More preferably, the differentiallydetectable labels will be of the same class (e.g., all radioactive, allfluorescent, etc.). Fluorescent labels are particularly preferred. Forexample, nucleotides can be labeled with FAM (emission at 518 nm), HEX(emission at 556 nm), Alexa 594 (emission at 612 nm) and Cy5 (emissionat 670 nm) to provide four differentially detectable nucleotides.

[0052] A large number of fluorescent nucleotide analogues are suitablefor use in the methods and compositions of this invention (see, e.g.,Kricka, L. J. (2002) “STAINS, LABLELS AND DETECTION STRATEGIES FORNUCLEIC ACIDS ASSAYS,” Ann. Clin. Biochem. 39:114-129). Suitablefluorescent labels include FAM (e.g., 6-FAM, etc.), HEX, Cy5, Cy5.5,Cy3, JOE, TAMRA (e.g., 6-TAMRA, 5-TAMRA, etc.), MANT, BODIPY (e.g.,BODIPY FL-14, BODIPY TR-14, BODIPY TMR-14, BODIPY R6G, etc.), Alexa(e.g., Alexa 430, Alexa 488, Alexa 546, Alexa 594, etc.), Texas Red(e.g., Texas Red-5, etc.), Cascade Blue, Fluorescein (e.g.,Fluorescein-12, etc.), TET (e.g., Tetramethylrhodamine-6, etc.),rhodamine (e.g., rhodamine red, rhodamine green, rhodamine 6G and ROX(e.g., 6-ROX, etc.). Rhodamine 110; rhodol; cyanine; coumarin or afluorescein compound (rhodamine 110, rhodol, or fluorescein compoundsthat have a 4′ or 5′ protected carbon) may be employed. Preferredexamples of such compounds include 4′(5′)thiofluorescein,4′(5′)-aminofluorescein, 4′(5′)-carboxyfluorescein,4′(5′)-chlorofluorescein, 4′(5′)-methylfluorescein,4′(5′)-sulfofluorescein, 4′(5′)-aminorhodol, 4′(5′)-carboxyrhodol,4′(5′)-chlororhodol, 4′(5′)-methylrhodol, 4′(5′)-sulforhodol;4′(5′)-aminorhodamine 110, 4′(5′)-carboxyrhodamine 110,4′(5′)-chlororhodamine 110, 4′(5′)-methylrhodamine 110,4′(5′)-sulforhodamine 110 and 4′(5′)thiorhodamine 110. “4′(5′)” meansthat at the 4 or 5′ position the hydrogen atom on the carbon atom issubstituted with a specific organic group or groups as previouslylisted. A 7-Amino, or sulfonated coumarin derivative may likewise beemployed. Fluorescein-12-dUTP, Rhodamine-5-dUTP, and Coumarin-6-dUTP maybe employed.

[0053] Any modification sufficient to render the incorporated nucleotideresistant to exonuclease activity treatment may be employed. Preferredexonuclease activity-resistant derivatives will possess α-thio orα-P-borano groups.

[0054] The exonuclease activity treatment degrades the target moleculesfrom their 3′ termini, and results in the creation of a set of targetmolecule fragments having nested 3′ termini. The polymerase activitytreatment results in the installation of an exonucleaseactivity-resistant chain-terminating nucleotide at this terminus. Thus,the net consequence of the exonuclease activity/polymerase activityreactions is the creation of a nested set of target molecule fragmentshaving a labeled exonuclease activity-resistant chain-terminatingnucleotide or nucleotide analog at their 3′ termini.

[0055] Once such a nested set of target molecule fragments has beenproduced, its members can be retrieved and analyzed to determine theidentity of the 3′ terminal nucleotides. For example, the molecules canbe subjected to gel electrophoresis. The label (of the incorporatedexonuclease activity-resistant chain-terminating nucleotide) associatedwith a particular band in the gel identifies the 3′ terminal nucleotidepresent in the molecules that make up that band. By comparing multiplebands, the sequence of the original target molecule can be readilydeduced. Although such analysis may be done manually, it is preferableto employ an automated sequencer for this purpose. The CEQ200XL andCEQ8000 Genetic Analysis Systems (Beckman-Coulter, Inc.) areparticularly preferred, especially in concert with the Biomek® 2000Laboratory Automation Workstation (Beckman-Coulter, Inc.). Although theuse of electrophoresis is a preferred method for determining thesequence of the labeled molecules, other methods, such as massspectroscopy, laser desorption mass spectrometry (LDMS), MALDI-TOF MS,hybridization to ordered arrays, flow cytometry, micro-chi[separation,etc. (Dovichi, N. J. et al. (2001) “DNA SEQUENCING BY CAPILLARY ARRAYELECTROPHORESIS,” Methods Molec. Biol. 167:225-39; Huber, C. G. et al.(2001) “ANALYSIS OF NUCLEIC ACIDS BY ON-LINE LIQUID CHROMATOGRAPHY-MASSSPECTROMETRY,” Mass Spectrom. Rev. 20:310-343; Buchholz, B. A. et al.(2001) “THE USE OF LIGHT SCATTERING FOR PRECISE CHARACTERIZATION OFPOLYMERS FOR DNA SEQUENCING BY CAPILLARY ELECTROPHORESIS,”Electrophoresis 22:4118-28; Mitnik, L. et al. (2001) “RECENT ADVANCES INDNA SEQUENCING BY CAPILLARY AND MICRODEVICE ELECTROPHORESIS,”Electrophoresis 22:4104-17; Bonk, T. et al. (2001) “MALDI-TOF-MSANALYSIS OF PROTEIN AND DNA,” Neuroscientist 7:6-12; Gawron, A. J. etal. (2001) “MICROCHIP ELECTROPHORETIC SEPARATION SYSTEMS FOR BIOMEDICALAND PHARMACEUTICAL ANALYSIS,” Eur. J. Pharm. Sci. 14(1):1-12).

[0056] The preferred embodiments of the present invention thus enablemultiple sequencing reactions (i.e., reactions involving theincorporation of different nucleotide species to be performedsimultaneously in a single reaction vessel. In its preferredembodiments, the invention differs from conventional dideoxynucleotidesequencing in that it can be conducted in the absence or substantialabsence of non-chain termination nucleotide triphosphates. In preferredembodiments of the invention, thermostabile polymerase activities arenot required and the use of modified polymerases can be minimized oravoided. Additionally, thermocycling is not required (thereby obviating“heated lid” or evaporation issues that affect conventionaldideoxynucleotide sequencing, while providing more rapid sequencing withhigher throughput). Additionally, in preferred embodiments of thepresent invention, the denaturation of template, in order for primer togain access to the template, is unnecessary.

[0057] In a preferred embodiment, the methods of the present inventionpermit the sequencing of both strands of a double-stranded nucleictarget molecules. In a further preferred embodiment, one strand of theproduced nested set of labeled oligonucleotides will additionally bespecially modified so as to facilitate their recovery and analysis. Inyet another preferred embodiment, such modification is accomplished bymodifying the target molecule to contain a haptenic group. Such amodification permits the oligonucleotides to be preferentially recoveredand/or immobilized by “agents” that bind to the haptenic group. Suchmodification may be introduced at any region of the target molecule, butwill preferably be provided at a site at or near the target molecule's5′ terminus. Suitable haptenic groups may be biotin groups, antigens,binding ligands, etc., where the “agent” is avidin (or streptavidin,etc.), or an antibody, receptor, or binding partner that preferentiallybinds to the employed haptenic group. In a further preferred embodiment,such modification is achieved by forming the target molecule from thetemplate-mediated extension of a primer molecule whose 5′ terminus hasbeen modified with the haptenic group.

[0058] In a particularly preferred embodiment, a preparation of asingle-stranded target nucleic acid molecule is prepared having a biotinmoiety at its 5′ terminus. The preparation is incubated in the presenceof an exonuclease activity (e.g., E. coli Exonuclease III) and apolymerase activity (e.g., Klenow polymerase), and four differentiallydetectable, exonuclease activity-resistant, chain-terminatingnucleotides under conditions sufficient to permit the exonucleaseactivity and polymerase activity reactions to proceed.

[0059] Reagents (such as EDTA, base, etc.) are added in amountssufficient to terminate the reaction. The nucleic acid molecules arecaptured onto a streptavidin plate by incubating them in contact withthe plate under suitable conditions (e.g., 25° C. for 0.5 h withoccasional mixing). The plate is then washed with alkali (e.g., 0.1 MNaOH at 25° C. for 5 min), and is treated with formamide and heat (98%formamide containing 10 mM EDTA at 94° C. for 5 min.). The material isthen loaded onto a gel, and is subjected to gel electrophoresis. Theresulting bands are then analyzed to determine the identity of thelabeled 3′ terminator nucleotide in each band, thereby providing thenucleotide sequence of the target molecule.

[0060] In a preferred example of such embodiment, illustrated in FIG. 1,one strand of a double-stranded nucleic target molecules will possess abiotin moiety (preferably at a site at or near the target molecule's 5′terminus). The molecules can then be incubated in the presence of avidin(or more preferably streptavidin) that is preferably bound to a solidsupport. The target molecules can be recovered from such a support bytreatment (such as heat denaturation) and then analyzed, as by gelelectrophoresis to determine the identity of the incorporated labelednucleotide.

[0061] The invention further contemplates additional preferredembodiments of such a method in which sequencing of only one strand canbe accomplished. For example, exonuclease activity degradation of the 3′terminus of the strand hybridized to the biotin-labeled strand can besterically inhibited by incubating the double-stranded molecule in thepresence of avidin or streptavidin. The binding of avidin orstreptavidin to the biotin group inhibits the degradation of the 3′terminus of the opposite strand, and thereby enables exonucleaseactivity to be conducted only or preferentially on one strand.Equivalently, a hapten or antigen may be used in place of biotin, and anantibody specific for such hapten or antigen may be employed in lieu ofthe avidin or streptavidin to sterically block the 3′ terminus of theopposite strand from exonuclease-mediated degradation.

[0062]FIG. 2 illustrates one approach to such a preferred embodiment ofthe invention. Two primers (“primer A” and “primer B”) are employed toproduce a preparation of target molecule. Primer A is designed tocontain dUridine residue(s); primer B is designed to contain anoligoribonucleotide region. The preparation is divided and one aliquottreated with uracil DNA glycosylase; another aliquot is treated withRNAse or alkali. Uracil DNA glycosylase removes the dUridine base, butdoes not cleave the DNA backbone. Exonuclease activity (such as forexample the exonuclease activity of Exonuclease III) cleaves the abasicsite and thereby degrades the 5′ terminus of the primer A strand, thusexposing the 3′ terminus of the primer B strand. The RNAse or alkalitreatment degrades the 5′ terminus of the primer B strand, thus exposingthe 3′ terminus of the primer A strand. Since exonuclease III does notdegrade an exposed 3′ terminus, such action causes the primer B strandof the Uracil DNA glycosylase-treated preparation and the primer Astrand of the RNAse or alkali-treated preparation to be resistant toexonuclease action. Incubation in the presence of an exonucleaseactivity, a polymerase activity and differentially detectable,exonuclease activity-resistant, chain-terminating nucleotides ornucleotide derivatives thus permits the methods of the present inventionto sequence the primer A strand of the uracil DNA glycosylase-treatedpreparation and the primer B strand of the RNAse or alkali-treatedpreparation. Other enzymatic activities may optionally be added tofacilitate any or all of the above reactions.

[0063] In an alternative approach, the target molecule is formed throughthe extension of two primers in a reaction that includes the provisionof phophothioate nucleotides, which are resistant to exonucleaseactivity. Such a reaction leads to the incorporation of phophothioatenucleotides into both primers. One primer (“primer A”) would preferablycontain 4 phosphothioates toward its 3′ end. The other primer (“primerB”) would contain phosphothioates on its 5′ end. Addition of a 5′ to 3′exonuclease would degrade the all “normal” phophodiester 5′ ends(alternatively the molecules could be formed with phosphorthioates, andtreated with 2-iodoethanol and/or 2,3-epoxy-1-propanol to cleave thephosphorthioate nucleotides). The 5′ terminus of the strand primed fromprimer A would be degraded to expose the 3′ terminus of the otherstrand; the 5′ terminus of the strand primed from primer B would not bedegraded. Since a single-stranded 3′ terminus is not susceptible toExonuclease III activity, treatment with exonuclease would degrade the5′ terminus of the primer A strand, and thereby render the 3′ terminusof the primer B strand resistant to exonuclease degradation. Thus, onlythe primer A strand would be sequenced in the reactions of the presentinvention.

[0064] Equivalently, ribonucleotides (or a primer containing anoligo-ribonucleotide region) can be employed in lieu of phosphothioatenucleotides. In a preferred embodiment of such an approach, the targetmolecules are subjected to treatment with RNAse or alkali so as todegrade the ribonucleotide portions of the target. By employing a“primer A” containing ribonucleotides toward its 3′ end and a “primer B”containing ribonucleotides on its 5′ end, treatment with RNAse or alkaliwould degrade the 5′ terminus of the primer A strand, and thereby renderthe 3′ terminus of the primer B strand resistant to exonucleasedegradation. Only the primer A strand would be sequenced in thereactions of the present invention.

[0065] In a further embodiment, the target molecules can be formed fromthe extension of a pair of primers, one of which has a restriction sitenot contained elsewhere in the sequence of the target that, when cleavedgenerates a 3′ overhang. Treatment with the restriction endonucleasethat recognizes such site thus renders the strand possessing theoverhang resistant to sequencing in accordance with the methods of thepresent invention. In a further embodiment, the target molecules can beformed from the extension of a pair of primers, each having a uniquerestriction site not contained elsewhere in the sequence of the targetmolecule that, when cleaved generates a 3′ overhang. This embodimentspermits the two strands of the target molecule to be separatelysequenced, by treatment with one restriction endonuclease, sequencing ofthe exonuclease sensitive strand, treatment with the second restrictionendonuclease, and sequencing of the second strand.

[0066] The methods of the present invention may be used to sequence anynucleic acid molecules, including nucleic acid molecules of mammalianorigin (especially human, simian, canine, bovine, ovine, feline, androdent), of plant origin, or of bacterial or lower eukaryotic origin.The methods of the present invention may be used to sequence nucleicacid molecules of pathogens (including bacterial, yeast, fungal andviral pathogens).

[0067] The present invention also concerns compositions and kitsspecially adapted to facilitate the above described methods. Exemplarycompositions include preparations of nucleotides that lack conventional(non-chain terminating) nucleotides but contain four differentiallydetectable exonuclease resistant, chain terminator nucleotide species,primers containing modified nucleotides or regions that can be employedto produce desired target molecules, and reagents and enzymes adapted toact upon such primers to permit the sequencing of one strand of anucleic acid molecule.

[0068] The present invention also concerns apparati, such as automatedsequenators that have been specially adapted to conduct the methods ofthe present invention.

[0069] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application had beenspecifically and individually indicated to be incorporated by reference.The discussion of the background to the invention herein is included toexplain the context of the invention. Such explanation is not anadmission that any of the material referred to was published, known, orpart of the prior art or common general knowledge anywhere in the worldas of the priority date of any of the aspects listed above.

[0070] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A method for determining the sequence of a regionof one strand of a double-stranded nucleic acid target molecule, whereinsaid method comprises incubating said nucleic acid target molecule inthe presence of an exonuclease activity, a polymerase activity and fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species.
 2. The method of claim 1, wherein fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species are employed.
 3. The method of claim 2,wherein at least one of said four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species is fluorescentlylabeled.
 4. The method of claim 3, wherein said four differentiallydetectable, exonuclease activity-resistant, chain terminator nucleotidespecies are fluorescently labeled.
 5. The method of claim 1, whereinsaid double-stranded nucleic acid target molecule possesses only one 3′terminus that is a substrate for said exonuclease activity.
 6. Themethod of claim 5, wherein said double-stranded nucleic acid targetmolecule possesses a 3′ terminus that extends beyond the 5′ terminus ofthe opposite strand. 7 The method of claim 5, wherein saiddouble-stranded nucleic acid target molecule possesses a 3′ terminusthat is sterically blocked from exonuclease activity degradation.
 8. Themethod of claim 1, wherein both strands of said double-stranded nucleicacid target molecule possess a 3′ terminus that is a substrate for saidexonuclease activity.
 9. The method of claim 1, wherein one 5′ terminusof said double-stranded nucleic acid target molecule possesses ahaptenic group.
 10. The method of claim 9, wherein said haptenic groupis biotin.
 11. The method of claim 1, wherein both 5′ termini of saiddouble-stranded nucleic acid target molecule possess a haptenic group.12. The method of claim 11, wherein said haptenic group is biotin.
 13. Amethod for determining the nucleotide sequence of a region of adouble-stranded nucleic acid target molecule, wherein said methodcomprises the steps: (A) incubating a preparation of saiddouble-stranded target molecule in the presence of a 3′ to 5′exonuclease activity, wherein said double-stranded nucleic acid targetmolecule possess at least one 3′ terminus that is a substrate for saidexonuclease activity, wherein said incubation is conducted underconditions sufficient to permit said exonuclease activity to produce anested population of double-stranded nucleic acid target molecule havingat least one degraded 3′ termini; (B) incubating said nested populationof double-stranded nucleic acid target molecule in the presence of apolymerase activity and at least one detectably labeled, exonucleaseactivity-resistant, chain terminator nucleotide species, wherein saidincubation is conducted under conditions sufficient to permit saidpolymerase activity to mediate the template-dependent incorporation ofone of said nucleotide species onto the 3′ terminus of a nucleic acidtarget molecule whose 3′ terminus was degraded by said exonucleaseactivity; and (C) determining the identity of the differentiallydetectable, exonuclease activity-resistant, chain terminator nucleotidespecies incorporated onto said 3′ terminus at said selected region. 14.The method of claim 13, wherein said steps A and B are conductedsimultaneously, and wherein said conditions employed are sufficient topermit said exonuclease activity to degrade said substrate termini andsufficient to permit said polymerase activity to mediate saidtemplate-dependent incorporation of said nucleotide species.
 15. Themethod of claim 13, wherein four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species are employed.16. The method of claim 13, wherein at least one of said fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species is fluorescently labeled.
 17. The methodof claim 16, wherein said four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species arefluorescently labeled.
 18. The method of claim 13, wherein saiddouble-stranded nucleic acid target molecule possesses only one 3′terminus that is a substrate for said exonuclease activity.
 19. Themethod of claim 18, wherein said double-stranded nucleic acid targetmolecule possesses a 3′ terminus that extends beyond the 5′ terminus ofthe opposite strand.
 20. The method of claim 18, wherein saiddouble-stranded nucleic acid target molecule possesses a 3′ terminusthat is sterically blocked from exonuclease activity degradation. 21.The method of claim 13, wherein both strands of said double-strandednucleic acid target molecule possess a 3′ terminus that is a substratefor said exonuclease activity.
 22. The method of claim 13, wherein one5′ terminus of said double-stranded nucleic acid target moleculepossesses a haptenic group.
 23. The method of claim 22, wherein saidhaptenic group is biotin.
 24. The method of claim 13, wherein both 5′termini of said double-stranded nucleic acid target molecule possess ahaptenic group.
 25. The method of claim 24, wherein said haptenic groupis biotin.
 26. An in vitro composition comprising a double-strandednucleic acid target molecule, an exonuclease activity, a polymeraseactivity and four differentially detectable, exonucleaseactivity-resistant, chain terminator nucleotide species.
 27. The invitro composition of claim 13, wherein at least one of said fourdifferentially detectable, exonuclease activity-resistant, chainterminator nucleotide species is fluorescently labeled.
 28. The in vitrocomposition of claim 27, wherein said four differentially detectable,exonuclease activity-resistant, chain terminator nucleotide species arefluorescently labeled.
 29. The in vitro composition of claim 13, whereinat least one 5′ terminus of said double-stranded nucleic acid targetmolecule possesses a haptenic group.
 30. The in vitro composition ofclaim 29 wherein said haptenic group is biotin.
 31. The in vitrocomposition of claim 13, wherein both 5′ termini of said double-strandednucleic acid target molecule possesses a haptenic group.
 32. The invitro composition of claim 31, wherein said haptenic group is biotin.33. A kit specially adapted to facilitate the sequencing of a targetnucleic acid molecule, said kit comprising a first container comprisinga primer A, a second container comprising a primer B, and a thirdcontainer containing an exonuclease activity, wherein said primers A andB mediate the amplification of a double-stranded nucleic acid moleculecomprising said target nucleic acid molecule, and wherein at least oneof said primer A or said primer B possesses a 5′ terminus having atleast one modified nucleotide.
 34. The kit of claim 33, wherein saidmodified nucleotide is a ribonucleotide, a dUridine nucleotide, aphosphothioate nucleotide, or a biotin-derivatized nucleotide.
 35. Thekit of claim 33, wherein said kit further comprises a fourth containercontaining four detectably labeled, exonuclease activity-resistant,chain terminator nucleotide species.
 36. The kit of claim 35, whereinsaid four detectably labeled, exonuclease activity-resistant, chainterminator nucleotide species are fluorescently labeled.
 37. Asequenator, comprising an apparatus for determining the identity offluoresecently labeled exonuclease activity-resistant, chain terminatornucleotide species incorporated onto the 3′ termini of a nucleic acidtarget molecule whose 3′ terminus was degraded by said exonuclease; andthen extended by a template-dependent polymerase to incorporate saidfluorescently labeled nucleotide species.